J. Agric. Food Chem. 2006, 54, 9017−9023
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Quantitative Analysis of Tylosin in Eggs by High Performance Liquid Chromatography with Electrospray Ionization Tandem Mass Spectrometry: Residue Depletion Kinetics after Administration via Feed and Drinking Water in Laying Hens GERD HAMSCHER,*,† SASITHORN LIMSUWAN,†,‡ NATTHASIT TANSAKUL,‡ MANFRED KIETZMANN‡
AND
Institute for Food Toxicology, University of Veterinary Medicine Hannover, Foundation, Bischofsholer Damm 15, D-30173 Hannover, Germany, and Department of Pharmacology, Toxicology and Pharmacy, University of Veterinary Medicine Hannover, Foundation, Buenteweg 17, D-30559 Hannover, Germany
Maximum residue limits (MRLs) have been established by the European Union when tylosin is used therapeutically. They are fixed at 200 µg/kg for eggs. A highly sensitive and selective quantitative liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) method suitable for monitoring tylosin residues in eggs to determine its depletion kinetics was developed and validated. For sample pretreatment all samples were liquid-liquid extracted with citrate buffer (pH 5.0) and acetonitrile. Liquid chromatographic separation was carried out on a reversed phase C18 column employing a 0.5% formic acid/acetonitrile gradient system. The tylosin recovery in eggs at a concentration range from 1.0-400 µg/kg was >82% with relative standard deviations between 1.5 and 11.0%. In two experimental studies administrating tylosin via feed (final dosage: 1.5 g/kg) or drinking water (final dosage: 0.5 g/L), no residues above the MRL were found during and after treatment. Moreover, all samples were well below the actual MRL of 200 µg/kg. Therefore, our residue data suggest that a withholding period for eggs is not required when laying hens are treated with tylosin in recommended dosages via feed or drinking water. KEYWORDS: Tylosin; residue; depletion; laying hen; withholding period; mass spectrometry
INTRODUCTION
Tylosin is a macrolide antibiotic frequently used in veterinary medicine with a high molecular weight of 916. The log kow value of tylosin is 1.63 (1), and thus the compound may be classified as a moderate hydrophilic veterinary drug. It has a macrocyclic ring structure containing 16 carbon lactones. This ring system is coupled to neutral and amino sugars, giving the molecule a basic character. The molecular structure of tylosin is shown in Figure 1. In general, macrolides (e.g. tylosin, erythromycin, oleandomycin) possess antimicrobial activity against gram-positive organisms andsin rare casessagainst gram-negative ones. They are nevertheless useful against mycoplasms, which have no cell wall. Macrolides inhibit protein synthesis by binding to the bacterial 50S ribosome, thus inhibiting polypeptide translation. Salts (tartrates, phosphates, lauryl sulfates) and esters (estolate, adipate, ethyl succinate) of the macrolides are synthesized to enhance their water solubility and/or bioavailability. After * Corresponding author. Tel.: (++49)(+511)-856-7784. Fax: (++49)(+511)-856-7680. E-mail:
[email protected]. † Institute for Food Toxicology. ‡ Department of Pharmacology, Toxicology and Pharmacy.
hydrolytic or enzymatic cleavage of the derivatives, the free bases are absorbed, and accumulate preferably in tissue. However, the oral bioavailability of macrolides depends on individual differences and on feed consumption, and is in the range of approximately 9% to 45% (2). Tylosin was previously reevaluated by the Committee for Veterinary Medicinal Products (CVMP). The committee considered a microbiological acceptable daily intake (ADI) of 0.360 mg per person (3). Finally, CVMP recommended the inclusion of tylosin in Annex I of Council Regulation (EEC) No. 2377/ 90. The maximum residue limits (MRLs) for the marker residue tylosin in all food producing species were as follows: muscle (100 µg/kg), fat (100 µg/kg), liver (100 µg/kg), kidney (100 µg/kg), milk (50 µg/kg), and eggs (200 µg/kg) (4). However, the maximum daily intake of tylosin based on these MRLs values will represent 80% of the ADI (3). The aims of the current study were to develop and validate an appropriate quantitative analytical method suitable for monitoring tylosin residues in whole eggs and to determine its depletion kinetics after oral administration via feed and drinking water.
10.1021/jf062205i CCC: $33.50 © 2006 American Chemical Society Published on Web 10/31/2006
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J. Agric. Food Chem., Vol. 54, No. 24, 2006
Hamscher et al.
Figure 1. Molecular structure of tylosin.
MATERIALS AND METHODS Hens and Experimental Design. Eighteen laying hens, 20 weeks old and weighing 2.0 (1.7-2.4) kg, were individually kept in cages, fed with a basal diet (Deuka all-mash L B 300/62; Du¨sseldorf, Germany), and given tap water ad libitum. They were divided into two groups of nine hens each. Tylosin 25% (Lilly, ELANCO Division, Bad Homburg, Germany), which consists of 25 g tylosin phosphate per 100 g, was mixed into their basal diet to obtain a final tylosin concentration of 1.5 g/kg. Tylan soluble (Lilly, ELANCO Division, Bad Homburg, Germany), which consists of 100% tylosin tartrate, was diluted in tap water to a final tylosin concentration of 0.5 g/L. The dosages were chosen according to the recommendations of the manufacturer. The drug was administered via feed and drinking water for five consecutive days. Food and water consumption during the study was recorded individually for each hen (see Tables 1 and 2). Eggs were collectedswhenever possiblesat 1 day intervals over a period of 15 days. The whole egg was immediately separated from the shell, weighed, and stored in individual polyethylene tubes with caps (84 × 30 mm, Sarstedt, Nu¨mbrecht, Germany) and frozen at -20 °C until they were analyzed for tylosin residues. Chemicals and Reference Substance. Acetonitrile (J.T. Baker, Griesheim, Germany) was of HPLC grade, and ammonium acetate, citric acid, formic acid, and sodium hydroxide (all obtained from Merck, Darmstadt, Germany) were of analytical reagent grade. Water was prepared in-house by a Milli-Q-System (Millipore, Eschborn, Germany) with a resistance of >18.2 MΩ/cm. Tylosin tartrate was obtained from Sigma (Munich, Germany) and had a purity of 90.8% (confirmed by Sigma employing HPLC analysis). A stock solution of tylosin was prepared by dissolving 1 mg of the drug in 1 mL methanol. The stock
solution was stored at -20 °C and was stable for at least 2 months. Working dilutions were prepared freshly on the day of use. For that purpose, prediluted methanolic solutions of 10 µg/mL, 1 µg/mL, and 0.1 µg/mL were prepared. From these solutions calibration standards from 0.00125 µg/mL to 1.25 µg/mL were prepared independantly in methanol. Extraction of Eggs. The extraction procedure was developed under consideration of a previously published method for the determination of various tetracyclines in whole eggs (5). Whole egg was homogenized using an Ultra Turrax homogenizer (T25 basic, IKA Labortechnik, Staufen, Germany). One gram of the homogenate was mixed with 0.6 mL of 1 M citrate buffer (pH 5) in 25 mL plastic tubes (Beckman, Munich, Germany) for 1 min using a magnetic stirrer. Ten milliliters of acetonitrile (HPLC gradient grade, J.T.Baker, Gross-Gerau, Germany) was then added, and the whole mixture was stirred for another 10 min. The sample was centrifuged for 10 min at approximately 11000g. The resulting supernatant was transferred into a 250 mL round-bottom glass flask, and the residue was mixed with 0.6 mL of distilled water and homogenized. Another 10 mL of acetonitrile was added to the sample, and the extraction procedure was repeated. The supernatants were combined and rotoevaporated to dryness under vacuum at 40 °C. It is highly recommended to use 250 mL flasks because the egg extract may foam heavily during evaporation. Furthermore, a continuous observation of the evaporation process should be performed. The dried sample was reconstituted with 400 µL methanol under sonification, transferred with a glass Pasteur pipet into a 1.5 mL plastic tube (Eppendorf, Germany), and centrifuged for 1 min at 20000g. The supernatant was transferred into a 1.5 mL autosampler vial (Thermo Electron Cooperation, Dreieich, Germany) equipped with a 300 µL insert (Chromatography Service, Langerwehe, Germany). Prior to LC/ MS/MS analysis, the samples were kept at 10 °C in the autosampler. Liquid Chromatography/Tandem Mass Spectrometry and Quantification. The LC/MS/MS procedure was developed under consideration of a previously published method for the determination of tylosin in soil and water (6). The HPLC system used was a gradient system consisting of a Thermoquest P4000 pump, an AS3000 autosampler (San Jose, CA), and a Puresil C18 column (5 µm, 4.6 × 150 mm, Waters Corporation, Milford, CT) operated at 23 °C. The flow of 1 mL/min was split 1:10 before entrance into the mass spectrometer. The mobile phase consisted of 0.5% formic acid in water with 1 mM ammonium acetate (solvent A, pH 2.5) and acetonitrile (solvent B). The gradient run was 0-50% B in 9 min, then held at 50% B for 1 min. After each run, the column was rinsed for 3 min with 99% acetonitrile and reequilibrated for 8 min with solvent A. The injection volume was 8 µL. The autosampler was rinsed after each injection with 3 mL of 90% methanol/10% 10 mM oxalic acid in water. Mass spectrometry was carried out using a LCQ ion trap with an electrospray ionization source (Finnigan Mat, San Jose, CA). A standard solution of tylosin (10 ng/µL) was infused through an integrated syringe pump at a flow rate of 10 µL/min for tuning the mass spectrometer and optimizing capillary temperature, sheath gas, and auxiliary gas flow rates. The source polarity was set positive, the spray needle voltage was 5 kV. Drying gas was nitrogen generated from pressurized air in an Ecoinert 2 ESP nitrogen generator (DWT-GmbH, Gelsenkirchen,
Table 1. Water Consumption of the Hens in Milliliters per Day During Treatment with Tylan Solublea water consumption [mL per day]
a
total tylosin consumption [mg]
hen [no.]
1b
2b
3b
4b
5b
1−5b
W1 W2 W3 W4 W5 W6 W7 W8 W9
300 300 300 430 430 430 300 300 300
270 270 270 370 370 370 270 270 270
300 220 210 230 260 600 210 20 260
290 190 160 200 260 490 260 20 240
350 200 200 250 290 380 220 20 260
755 590 570 740 805 1135 630 315 665
Final tylosin dosage: 0.5 g/L. b Day(s) of treatment.
J. Agric. Food Chem., Vol. 54, No. 24, 2006
Detection of Tylosin in Whole Eggs by LC/MS/MS
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Table 2. Feed Consumption of the Hens in Grams per Day During Treatment with Tylan 25%a food consumption [g per day]
a
total tylosin consumption [mg]
hen [no.]
1b
2b
3b
4b
5b
1−5b
F1c F2 F3 F4 F5 F6 F7 F8 F9
15 130 120 105 145 155 140 120 135
15 130 130 120 140 130 120 130 120
15 115 110 115 130 130 130 125 120
35 110 95 130 125 135 120 120 130
35 85 95 90 120 130 115 130 115
173 855 825 840 990 1020 938 938 930
Final tylosin dosage: 1.5 g/kg feed. b Day(s) of treatment. c Hen F1 was sick during treatment thus leading to a decreased food consumption.
Table 3. Recoveries (n ) 3) at Various Concentrations and Day-to-Day Variation at the 50 µg/kg Level (n ) 7) and the Corresponding Relative Standard Deviations (RSD) of Tylosin in Whole Egg
Table 4. Tylosin Concentrations [µg/kg] in Whole Eggs Laid by Hens Fed a Diet Containing 1.5 g/kg Tylosin or Receiving Drinking Water Containing 0.5 g/L Tylosina tylosin [µg/kg]
concn [µg/kg] 1 5 10 20 50a 100 200 400 a
recovery [%]
RSD [%]
102.1 ± 3.4 82.0 ± 9.0 104.2 ± 5.4 88.8 ± 1.3 92.5 ± 8.8 88.3 ± 7.5 105.1 ± 7.0 110.9 ± 3.9
3.3 11.0 5.2 1.5 9.5 8.5 6.7 3.5
hen [no.]
day 0 (control) Xc
day 5
W1 W2 W3 W4 W5 W6
